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1.  Direct inhibitory effects of Ganciclovir on ICAM-1 expression and proliferation in human coronary vascular cells (SI/MPL-ratio: >1) 
Summary
Background
Treatment of the human cytomegalovirus (HCMV) infection with ganciclovir has beneficial indirect effects on the complex interactions of HCMV with restenosis, atherosclerosis, and transplant vascular sclerosis. The current study reports on direct effects of ganciclovir on expression of ICAM-1 and cell proliferation, key events of coronary atherosclerosis/restenosis. A potential clinical relevance of the data will be evaluated with the help of SI/MPL-ratio’s.
Material/Methods
Definition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (ganciclovir: 9 μg/ml). Part I of the study investigated in cytoflow studies the effect of ganciclovir (0.05–5000 μg/mL) on TNF-a induced expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells derived from umbilical veins (HUVEC), human coronary endothelial cells (HCAEC), and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of ganciclovir (0.05–5000 μg/mL) on cell proliferation (HUVEC, HCAEC, and HCMSMC). In part III cytotoxic effects of ganciclovir (0.05–5000 μg/mL) were studied (HUVEC, HCAEC, and HCMSMC).
Results
Ganciclovir caused slight but significant inhibitory effects on expression of ICAM-1 in HUVEC, HCAEC, and HCMSMC. In all three cell types studied strong dose depending significant antiproliferative effects of ganciclovir were detected. Partially, the antiproliferative effects of ganciclovir were caused by cytotoxic effects.
Conclusions
SI/MPL-ratio’s >1 in HCAEC and HCMSMC indicate that the inhibitory effects of gancliclovir on ICAM-1-expression and cell proliferation may only be expected in vivo following local high dose administration e.g. in drug eluting stents (DES).
doi:10.12659/MSM.881310
PMCID: PMC3524678  PMID: 21169918
Ganciclovir; adhesion molecule ICAM-1; cell proliferation; Coronary Restenosis; drug eluting stents
2.  HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia 
BMC Microbiology  2007;7:68.
Background
The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.
Results
During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.
Conclusion
The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.
doi:10.1186/1471-2180-7-68
PMCID: PMC1950876  PMID: 17659083
3.  Sirolimus inhibits key events of restenosis in vitro/ex vivo: evaluation of the clinical relevance of the data by SI/MPL- and SI/DES-ratio's 
Background
Sirolimus (SRL, Rapamycin) has been used successfully to inhibit restenosis both in drug eluting stents (DES) and after systemic application. The current study reports on the effects of SRL in various human in vitro/ex vivo models and evaluates the theoretical clinical relevance of the data by SI/MPL- and SI/DES-ratio's.
Methods
Definition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (6.4 ng/ml for SRL). Definition of the SI/DES-ratio: relation between significant inhibitory effects in vitro/ex vivo and the drug concentration in DES (7.5 mg/ml in the ISAR drug-eluting stent platform). Part I of the study investigated in cytoflow studies the effect of SRL (0.01–1000 ng/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of SRL (0.01–1000 ng/ml) on cell migration of HCMSMC. In part III, IV, and V of the study ex vivo angioplasty (9 bar) was carried out in a human organ culture model (HOC-model). SRL (50 ng/ml) was added for a period of 21 days, after 21 and 56 days cell proliferation, apoptosis, and neointimal hyperplasia was studied.
Results
Expression of ICAM-1 was significantly inhibited both in HCAEC (SRL ≥ 0.01 ng/ml) and HCMSMC (SRL ≥ 10 ng/ml). SRL in concentrations ≥ 0.1 ng/ml significantly inhibited migration of HCMSMC. Cell proliferation and neointimal hyperplasia was inhibited at day 21 and day 56, significance (p < 0.01) was achieved for the inhibitory effect on cell proliferation in the media at day 21. The number of apoptotic cells was always below 1%.
Conclusion
SI/MPL-ratio's ≤ 1 (ICAM-1 expression, cell migration) characterize inhibitory effects of SRL that can be theoretically expected both after systemic and local high dose administration, a SI/MPL-ratio of 7.81 (cell proliferation) represents an effect that was achieved with drug concentrations 7.81-times the MPL. SI/DES-ratio's between 10-6 and 10-8 indicate that the described inhibitory effects of SRL have been detected with micro to nano parts of the SRL concentration in the ISAR drug-eluting stent platform. Drug concentrations in DES will be a central issue in the future.
doi:10.1186/1471-2261-7-15
PMCID: PMC1878500  PMID: 17498286
4.  Edge restenosis: impact of low dose irradiation on cell proliferation and ICAM-1 expression 
Background
Low dose irradiation (LDI) of uninjured segments is the consequence of the suggestion of many authors to extend the irradiation area in vascular brachytherapy to minimize the edge effect. Atherosclerosis is a general disease and the uninjured segment close to the intervention area is often atherosclerotic as well, consisting of neointimal smooth muscle cells (SMC) and quiescent monocytes (MC). The current study imitates this complex situation in vitro and investigates the effect of LDI on proliferation of SMC and expression of intercellular adhesion molecule-1 (ICAM-1) in MC.
Methods
Plaque tissue from advanced primary stenosing lesions of human coronary arteries (9 patients, age: 61 ± 7 years) was extracted by local or extensive thrombendarterectomy. SMC were isolated and identified by positive reaction with smooth muscle α-actin. MC were isolated from buffy coat leukocytes using the MACS cell isolation kit. For identification of MC flow-cytometry analysis of FITC-conjugated CD68 and CD14 (FACScan) was applied. SMC and MC were irradiated using megavoltage photon irradiation (CLINAC2300 C/D, VARIAN, USA) of 6 mV at a focus-surface distance of 100 cm and a dose rate of 6 Gy min-1 with single doses of 1 Gy, 4 Gy, and 10 Gy. The effect on proliferation of SMC was analysed at day 10, 15, and 20. Secondly, total RNA of MC was isolated 1 h, 2 h, 3 h, and 4 h after irradiation and 5 μg of RNA was used in standard Northern blot analysis with ICAM-1 cDNA-probes.
Results
Both inhibitory and stimulatory effects were detected after irradiation of SMC with a dose of 1 Gy. At day 10 and 15 a significant antiproliferative effect was found; at day 20 after irradiation cell proliferation was significantly stimulated. Irradiation with 4 Gy and 10 Gy caused dose dependent inhibitory effects at day 10, 15, and 20. Expression of ICAM-1 in human MC was neihter inhibited nor stimulated by LDI.
Conclusion
Thus, the stimulatory effect of LDI on SMC proliferation at day 20 days after irradiation may be the in vitro equivalent of a beginning edge effect. Extending the irradiation area in vascular brachytherapy in vivo may therefore merely postpone and not inhibit the edge effect. The data do not indicate that expression of ICAM-1 in quiescent MC is involved in the process.
doi:10.1186/1471-2261-6-32
PMCID: PMC1526455  PMID: 16827927
5.  Effects of abciximab on key pattern of human coronary restenosis in vitro: impact of the SI/MPL-ratio 
Background
The significant reduction of angiographic restenosis rates in the ISAR-SWEET study (intracoronary stenting and antithrombotic regimen: is abciximab a superior way to eliminate elevated thrombotic risk in diabetes) raises the question of whether abciximab acts on clopidogrel-independent mechanisms in suppressing neointimal hyperplasia. The current study investigates the direct effect of abciximab on ICAM-1 expression, migration and proliferation.
Methods
ICAM-1: Part I of the study investigates in cytoflow studies the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1). Migration: Part II of the study explored the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on migration of HCMSMC over a period of 24 h. Proliferation: Part III of the study investigated the effect of abciximab (0.0002, 0.002, 0.02, 0.2, 2.0, and 20.0 μg/ml) on proliferation of HUVEC, HCAEC, and HCMSMC after an incubation period of 5 days.
Results
ICAM-1: In human venous endothelial cells (HUVEC), human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC) no inhibitory or stimulatory effect on expression of ICAM-1 was detected. Migration: After incubation of HCMSMC with abciximab in concentrations of 0.0002 – 2 μg/ml a stimulatory effect on cell migration was detected, statistical significance was achieved after incubation with 0.002 μg/ml (p < 0.05), 0.002 μg/ml (p < 0.001), and 0.2 μg/ml (p < 0.05). Proliferation: Small but statistically significant antiproliferative effects of abciximab were detected after incubation of HUVEC (0.02 and 2.0 μg/ml; p = 0.01 and p < 0.01), HCAEC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0,01), and HCMSMC (2.0 and 20.0 μg/ml; p < 0.05 and p < 0.05). The significant inhibition (SI) of cell proliferation found in HCAEC and HCMSMC was achieved with drug concentrations more than 10 times beyond the maximal plasma level (MPL), resulting in a SI/MPL-ratio > 1.
Conclusion
Thus, the anti-restenotic effects of systemically administered abciximab reported in the ISAR-SWEET-study were not caused by a direct inhibitory effect on ICAM-1 expression, migration or proliferation.
doi:10.1186/1471-2261-6-14
PMCID: PMC1475639  PMID: 16595000
6.  Effects of mycophenolate mofetil on key pattern of coronary restenosis: a cascade of in vitro and ex vivo models 
Background
Mycophenolate mofetil (MMF), the prodrug of mycophenolic acid (MPA), is a rationally designed immunosuppressive drug. The current study investigates the effect of MMF on key pattern of restenosis in a cascade of in vitro and ex vivo models.
Methods
Part I of the study investigated in northern blot and cytoflow studies the effect of MMF (50, 100, 150, 200, 250, and 300 μg/mL) on TNF-α induced expression of intercellular adhesion molecule 1 (ICAM-1) in human coronary endothelial cells (HCAEC) and human coronary medial smooth muscle cells (HCMSMC). Part II of the study applied a human coronary 3D model of leukocyte attack, the 3DLA-model. HCAEC and HCMSMC were cultured on both sides of a polycarbonate filters, mimicking the internal elastic membrane. Leukocyte attack (LA) was carried out by adding human monocytes (MC) on the endothelial side. The effect of MMF (50 μg/mL) on adhesion and chemotaxis (0.5, 1, 2, 3, 4, 6, and 24 h after LA) and the effect on proliferation of co-cultured HCMSMC (24 h after LA) was studied. In part III of the study a porcine coronary organ culture model of restenosis (POC-model) was used. After ex vivo ballooning MMF (50 μg/mL) was added to the cultures for a period of 1, 2, 3, 4, 5, 6, and 7 days. The effect on reactive cell proliferation and neointimal thickening was studied at day 7 and day 28 after ballooning.
Results
Expression of ICAM-1 in northern blot and cytoflow studies was neither clearly inhibited nor stimulated after administration of MMF in the clinical relevant concentration of 50 μg/mL. In the 3DLA-model 50 μg/mL of MMF caused a significant antiproliferative effect (p < 0.001) in co-cultured HCMSMC but had no effect on MC-adhesion and MC-chemotaxis. In the ex vivo POC-model neighter reactive cell proliferation at day 7 nor neointimal hyperplasia at day 28 were significantly inhibited by MMF (50 μg/mL).
Conclusion
Thus, the data demonstrate a significant antiproliferative effect of clinical relevant levels of MMF (50 μg/mL) in the 3DLA-model. The antiproliferative effect was a direct antiproliferative effect that was not triggered via reduced expression of ICAM-1 or via an inhibition of MC-adhesion and chemotaxis. Probably due to technical limitations (as e.g. the missing of perfusion) the antiproliferative effect of MMF (50 μg/mL) could not be reproduced in the coronary organ culture model. A cascade of focused in vitro and ex vivo models may help to gather informations on drug effects before large experimental studies are initiated.
doi:10.1186/1471-2261-5-9
PMCID: PMC1156877  PMID: 15890069
7.  Simultaneous intra/extravascular administration of antiproliferative agents as a new strategy to inhibit restenosis: The peak of reactive cell proliferation as a hallmark for the duration of the treatment 
Background
Strictly intravascular approaches for the treatment of postangioplasty restenosis are effective in the intima and the inner parts of the media but may be insufficient to control redundant pathways in the more outer parts of the media and the adventitia. An inverse situation may occur subsequently to a strictly extravascular approach, like the recently suggested pericardial approach in pigs. We hypothesized that simultaneous intra/extravascular administration of anti-restenotic agents inhibits restenosis by blocking all stimulatory pathways in the entire arterial wall.
Methods
Fresh hearts of 25 domestic pigs were obtained from a local slaughterhouse. Left anterior descending coronary arteries (LAD) were harvested, cut into cylindric 5 mm segments, and cultured as ex vivo porcine organ cultures (POCs). After 9 bar ballooning simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) was carried out for a period of 1, 2, 3, 4, 5, 6, and 7 days. At day 7 and 28 proliferative activity (BrdU), neointimal thickening, and staining against smooth muscle α-actin and vWF was analysed.
Results
7 days after ballooning administration of diltiazem for 4, 5, 6, and 7 days inhibited reactive cell proliferation by more than 50% (n.s.) as compared to control, 28 days after ballooning administration for 6 and 7 days inhibited neointimal thickening by more than 75% (p < 0.05). Simultaneous intra/extravascular administration of high dose diltiazem did not affect the expression of vWF in endothelial cells or smooth muscle α-actin in smooth muscle cells.
Conclusions
Simultaneous intra/extravascular administration of high dose diltiazem (50 μg/mL) has to be maintained for at least 6 days to achieve a significant inhibition of neointimal thickening. The data demonstrate the importance of the maximal reactive cell proliferation (= day 7 in the POC-model) for the calculation of the duration of the treatment period.
PMCID: PMC65511  PMID: 11825339
8.  Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells 
Background
Activation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50).
Results
Smooth muscle cells (SMC) from human coronary plaque material (HCPSMC, plaque material of 52 patients), SMC from the human coronary media (HCMSMC), human endothelial cells (EC) from umbilical veins (HUVEC), and human coronary EC (HCAEC) were successfully isolated (HCPSMC, HUVEC), identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC). 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs) RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM) and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50.
Conclusions
The data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.
doi:10.1186/1471-2199-2-7
PMCID: PMC48145  PMID: 11532196

Results 1-8 (8)